Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin.

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Iron uptake mechanisms as key virulence factors in bacterial fish pathogens.

This review summarizes the current knowledge about iron uptake systems in bacterial fish pathogens and their involvement in the infective process. Like most animal pathogens, fish pathogens have evolved sophisticated iron uptake mechanisms some of which are key virulence factors for colonization of the host. Among these systems, siderophore production and heme uptake systems are the best studied in fish pathogenic bacteria. Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens as key virulence factors to cause disease in fish. In many other bacterial fish pathogens production of siderophores was demonstrated but the compounds were not yet chemically characterized and their role in virulence was not determined. The role of heme uptake in virulence was not yet clearly elucidated in fish pathogens although there exist evidence that these systems are expressed in fish tissues during infection. The relationship of other systems, like Fe(II) transporters or the use of citrate as iron carrier, with virulence is also unclear. Future trends of research on all these iron uptake mechanisms in bacterial fish pathogens are also discussed.

Virulence properties of three new Photobacterium species affecting cultured fish.

AIMS: Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photobacterium malacitanum and Photobacterium andalusiense associated with diseases of cultured redbanded seabream (Pagrus auriga) were studied. The exoenzymatic activities, adherence and cytotoxic capabilities, and iron-uptake mechanisms were determined both in bacterial extracellular products (ECP) and whole bacterial cells. The histopathology damages provoked on redbanded seabream by the ECP was also studied. METHODS AND RESULTS: The highest exoenzymatic activities of the ECP were alkaline- and acid-phosphatase, phosphohydrolase and lipase. The ECP were strongly lethal for fish at 4-96 h post-inoculation (p.i). Histological changes were evident at 96 hpi of ECP, affecting head kidney, splenic parenchyma and heart. Cytotoxicity assays, on three fish lines and one human cell line, were conducted using whole bacterial cells and their ECP. The new species tested were cytotoxic only for fish cell lines using whole bacterial cells. Bacterial adherence showed an adherence index moderate on CHSE-214 cell line. All strains showed variable haemolytic activity, and were able to grow under iron-limiting conditions, although the CAS reactivitiy was very low. However, all strains produced high amounts of extracelullar citrate that could be used as iron carrier, and use haem as iron source, except the P. toruni strains because a deletion in the genomic region encoding this ability in all Vibrionaceae members. CONCLUSIONS: The toxic activity of the bacterial ECPs was thermolabile, and not associated with their thermoresistant lipopolysaccharide content. The virulence of the strains tested could not be related to the haemolytic activity. Iron uptake could be based on the use of endogenous citrate as iron carrier and P. toruni lacks the ability to use haem as iron source. SIGNIFICANCE AND IMPACT OF THE STUDY: The study analyses for the first time the virulence properties of three new species of Photobacterium pathogenic for fish.

Maraviroc contributes to the restoration of the homeostasis of regulatory T-cell subsets in antiretroviral-naive HIV-infected subjects.

Regulatory T (Treg) cells comprise different functional subsets with different CCR5 expression. Treg homeostasis is disrupted by HIV but the effect of treatment has barely been explored. In a longitudinal design, we compared the effect of a maraviroc-containing (n = 9) or sparing (n = 12) therapy in antiretroviral-naive HIV-positive participants on peripheral FoxP3(low) CD45RA(+) (nTreg), FoxP3(high) CD45RA(-) (eTreg) and FoxP3(low) CD45RA(-) (non-Treg) cells. Maraviroc significantly reduced all subsets in the short-term and, except for nTreg cells, also normalized them in the long-term. The correlation between eTreg cells and CD4 counts, lost before treatment, was only restored by maraviroc. The differential effect of maraviroc on Treg subsets contributes to understanding its immunomodulatory effects.

Specific patterns of CD4-associated immunosenescence in vertically HIV-infected subjects.

Vertical transmission of human immunodeficiency virus (HIV) represents an important world-wide health problem although the incidence in developed countries has been drastically reduced by the extensive use of highly active antiretroviral therapy. Vertically HIV-infected subjects have been exposed to the virus during the maturation of their immune systems and have suffered a persistent chronic activation throughout their lifetime; the consequences of this situation for their immune system are not fully understood. The objective of this study was to analyse immunosenescence-related parameters in different CD4 T-cell subsets. Fifty-seven vertically HIV-infected subjects and 32 age-matched healthy subjects were studied. Activation (HLA(-) DR(+) ), senescence (CD28(-) CD57(+) ) and proliferation (Ki67(+) ) were analysed on different CD4 T-cell subsets: naive (CD45RA(+) CD27(+) ), memory (CD45RO(+) CD27(+) ), effector memory (CD45RO(+) CD27(-) ) and effector memory RA (CD45RA(+) CD27(-) ). Compared with healthy subjects, vertically HIV-infected subjects showed increased naive and memory CD4 T-cell frequencies (p 0.035 and p 0.010, respectively) but similar frequencies of both effector subsets. Whereas naive CD4 T cells were not further altered, memory CD4 T cells presented increased levels of senescence and proliferation markers (p <0.001), effector memory CD4 T cells presented increased levels of activation, senescence and proliferation markers (p <0.001) and effector memory RA CD4 T cells presented increased levels of activation and senescence (p <0.001) compared with healthy subjects. Despite long periods of infection, vertically HIV-infected subjects show specific patterns of immunosenescence, revealing a preserved CD4 T-cell homeostasis for subset differentiation and distribution. Nevertheless, excepting the naive subpopulation, all subsets experienced some immunosenescence, pointing to uncertain consequences of the future aging process in these subjects.

Clips for the prevention and treatment of postpolypectomy bleeding (hemoclips in polypectomy).

INTRODUCTION: The most accurate method for the prevention and treatment of complications after polypectomy has not been well defined. The prophylactic use of hemoclips may reduce the risk of bleeding, mainly in pedunculated big polyps. OBJECTIVE: To evaluate the accuracy of hemoclips in the prophylaxis and treatment of bleeding after endoscopic polypectomy. MATERIAL AND METHODS: Retrospective study of 223 consecutive endoscopic polypectomies performed in our Endoscopy Unit between january and october 2001. Hemoclips were routinely used only for large polyps (15 to 40 mm); all of them were located in the colon except one, a gastric polyp. RESULTS: From a total of 223 polypectomies (215 patients), hemoclips were used for 34 (15.2%), in 30 of them just before and in 4 just after polypectomy. When used prophylactically no complication was observed, except one mild bleeding episode (3.3%) that stopped with the placing of a second hemoclip. The therapeutic clipping (4 polypectomies) induced immediate haemostasis in all cases. CONCLUSIONS: The prophylactic use of hemoclips is associated with a very low risk of bleeding after endoscopic resection of big polyps. Therapeutic clipping is an effective measure for polypectomy-related bleeding.

Clips en la prevención y tratamiento de la hemorragia post-polipectomía endoscópica (polipectomía con hemoclips) / Clips in the prevention and treatment of endoscopic polypectomyrelated bleeding (hemoclips in polypectomy)

Introducción: el método más efectivo en la prevención y tratamiento de complicaciones post-polipectomía endoscópica no ha sido bien definido. La colocación de clips es uno de los procedimientos utilizados. Su uso profiláctico, principalmente en pólipos pediculados de gran tamaño, puede reducir el riesgo de sangrado. Objetivo: evaluar la eficacia de los clips en la profilaxis y tratamiento del sangrado post-polipectomía endoscópica. Material y métodos: estudio retrospectivo de 223 polipectomías endoscópicas realizadas consecutivamente en nuestro servicio entre los meses de enero y octubre de 2001. Los clips se emplearon únicamente en grandes pólipos (de 15 a 40 mm); todos eran colónicos excepto uno gástrico. Resultados: de un total de 223 polipectomías (215 pacientes) se colocaron clips en 34 de ellas (15,2 por ciento). Se hizo de forma profiláctica en 30 polipectomías y terapéutica en 4. En las aplicaciones profilácticas no se registraron complicaciones salvo un caso de sangrado leve (3,3 por ciento) que cedió tras la colocación de un nuevo hemoclip. El clipaje terapéutico (4 polipectomías) supuso hemostasia inmediata en todos los casos. Conclusiones: la utilización de hemoclips constituye un procedimiento seguro y eficaz, tanto para la profilaxis como para el tratamiento del sangrado post-polipectomía, facilitando la resección en bloque, lo que mejora la evaluación histológica de la pieza (AU)

[Endoscopic percutaneous gastrostomy. Experience at a general hospital]. / Gastrostomía percutánea endoscópica. Experiencia en un hospital general.

Percutaneous endoscopic gastrostomy is a technique scantly used in Spanish hospitals. Its finality is the nutrition of patients with dysphagia, by avoiding the inconveniences and complications of transnasal feeding tubes. Our group has begun a protocol of endoscopically placed gastrostomies and in this work we present its first series; it includes 23 cases of patients with dysphagia secondary to neurological disorders. We have employed the original "pull technique" from Gauderer and Ponsky and we have used homemade gastrostomy tubes. Only two mild complications were observed (stoma infection). In our experience, this technique is easy and has few complications. Furthermore, if homemade tubes are used, the economic cost is very low. In this paper, beside of communicating our results, we summarize the percutaneous endoscopic gastrostomy technique and the way for constructing the tubes.

[Gastroesophageal infection caused by cytomegalovirus in immunodepressed patients. Presentation of 3 cases]. / Infección gastroesofágica por citomegalovirus en pacientes inmunodeprimidos. Presentación de tres casos.

We report three cases of upper gastrointestinal infection by Cytomegalovirus in immunocompromised patients. Two patients suffered from AIDS and the other one had a recent heart transplantation and was on immunodepressant drugs for an episode of rejection. The stomach in two cases and the esophagus in the other were the affected organs. In all cases there were upper gastrointestinal symptoms and the diagnosis was established by biopsy of endoscopically suspicious lesions. Antiviral therapy was effective in all three cases. This diagnosis has to be considered whenever immunocompromised patients present with unusual clinical and endoscopic findings.